Bispecific antibody capable of being combined with immune cells to enhance tumor killing capability, and preparation method therefor and application thereof

ABSTRACT

The present invention provides a bispecific antibody capable of being combined with immune cells to enhance a targeting tumor killing capability, and a preparation method therefor and an application thereof. Antibodies and degradable nanoparticles are connected by using a chemical method, so as to make the one nanoparticle be connected to two or more antibody molecules at the same time, wherein one antibody can be specifically bound with immune cells, and the other antibody or the other antibodies can be specifically bound to tumor cells so as to achieve the effect of enhancing the capability of the immune cells for specifically killing tumor cells in a targeting way.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/548,630, filed Aug. 3, 2017, which is a 371 of International PatentApplication No. PCT/CN2016/079307 filed on Apr. 14, 2016, the entirecontent and disclosure of each is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the field of immunotherapy, andparticularly to a bispecific antibody capable of being combined withimmune cells to enhance a targeting tumor killing capability, and apreparation method therefor and an application thereof.

BACKGROUND ART

In 2008, New England Journal of Medicine reported that one case ofimmunotherapy on advanced melanoma was successful, and the patient haddeveloped multiple metastases in the body, all of which disappearedafter autologous CD4+ T cell treatment, with 26 months of long-termsurvival via a follow-up. In 2010, FDA approved autologous immune celltherapy technology of Dendreon Corporation for the clinical applicationof prostate cancer, and in 2011, three scientists engaged in cancerimmunotherapy also was awarded Nobel Prize for medicine, suggesting abroad prospect of immunotherapy in malignant treatment.

In recent years, the use of genetically modified and induced T cellimmunotherapy has achieved good results on tumor, which points out a newdirection for developing immune cell therapy of tumor. This suggests abroad prospect of immunotherapy in malignant treatment.

The immune cell therapy technology has undergone the development of LAK,CIK, DC-CIK and now is rapidly growing toward antigen-loading DC-inducedT-cell (DiKat), genetically modified DC-induced T cells (AV/LV-DC-CTL)and genetically modified chimeric antigen receptor T cells (CAR-T).Currently, Novartis announced a result of B lymphoma clinical trial forthe CD19 target with a rate of complete efficacy of 93%, furtherhighlighting the prospect of the immune cell therapy of cancer. However,CAR-T is challenged because of its complex cell preparation process, aswell as high costs (the cost of each course is expected to be 500,000 USdollars), and currently shows a good efficacy only for CD19-positive Blymphoma, suggesting its obvious limitations.

An urgent need exists for solving the problem on how to develop animmune cell therapy technology, which has enhanced specificity andeffectiveness and the advantages of simple and convenient use etc.

Contents of Invention

The present invention provides a bispecific antibody, as well as apreparation method therefor and an application thereof, and particularlya bispecific antibody capable of being combined with immune cells toenhance a targeting tumor killing capability, as well as a preparationmethod therefor and an application thereof.

To this end, the present invention adopts the following technicalsolutions:

In a first aspect, the present invention provides a bispecific antibodycapable of being combined with immune cells to enhance a targeting tumorkilling capability, said bispecific antibody comprising a first antibodymoiety that binds to an antigen expressed on an effector T cell and asecond antibody moiety that binds to an antigen expressed on a targetcell.

The second antibody moiety described herein may be one or moreantibodies that can bind to the target cell.

In the present invention, the first antibody moiety and the secondantibody moiety are connected by a nanomaterial. The connection can bemade by using the carboxyl group of the nanoparticle surface and theamino group of the antibody.

A “multispecific antibody” is an antibody that may simultaneously bindto at least two targets with different structures (e.g., two differentantigens, two different epitopes on the same antigen, or a hapten and/orantigen or epitope). A “bispecific antibody” is an antibody that canbind to two targets with different structures simultaneously. The“multispecific antibody” or “bispecific antibody” described hereinincludes multiple or two antibodies (e.g., two monoclonal antibodies)that are connected by a nanomaterial and bind to different targets. Themultispecific antibody or bispecific antibody described herein may haveat least one antibody that specifically binds to a T cell and at leastone antibody that specifically binds to an antigen produced by adiseased cell, tissue, organ or pathogen, or an antigen related thereto(e.g., a tumor-associated antigen), which antibodies are connected by ananomaterial.

For the purpose of immune cell targeting therapy, the present inventionutilizes the specific binding ability of an antibody to attach theantibodies capable of specifically recognizing a tumor cell and a tumorkiller cell to a clinically available degradable nanomaterial, so as toform a bispecific antibody. In contrast, most anti-cancer monoclonalantibodies as a drug for the treatment of diseases are mainly based ontheir inherent biological functions, including complement-mediatedcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), apoptosis induction, opsonophagocytosis etc. The bispecificantibody of the invention not only specifically recognizes and binds toa tumor cell, but also recognizes and binds to a killer T lymphocyte,thereby assisting the T cell to recognize the tumor cell and narrowingthe physical distance between the tumor cell and the killer Tlymphocyte, and thus facilitating the killing of the tumor cell by the Tlymphocyte. Accordingly, the specificity and effectiveness of immunecell therapy are strengthened.

The nanomaterial described herein is a biodegradable nanomaterial.

The nanomaterial described herein is generally present in the form ofnanoparticle, which is selected from a conventional degradablenanomaterial, such as any of polylactic acid-glycolic acid, polylacticacid, polycaprolactone, polybutylene glycol succinate, polyaniline,polycarbonate, glycolide-lactide copolymer or glycolide-caprolactonecopolymer, or a mixture thereof, preferably, but not limited to these.

The nanomaterial used herein possesses the advantages of slow releasingand good biocompatibility etc. For example, PLGA is a pharmaceuticalexcipient approved by FDA, which has good biocompatibility andbiodegradability, no toxicity and irritation, high strength and iseasily processed and molded. It is finally decomposed into hydratedcarbon dioxide by enzymatically hydrolysis in vivo, and can becompletely absorbed within 3-6 months after being implanted into thebody.

In the present invention, the target cell is B cell, cancer cell, orpathogen cell, etc.

Preferably, the antigen expressed on the target cell is any one ofcarbonic anhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3, A33antibody-specific antigen, ANG2, ART-4, B7, Ba 733, BAGE, BrE3-antigen,CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46,CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a,CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154,CDC27, CDK-4/m, CDKN2A, CS1, CXCR4, CXCR7, CXCL12, HIF-1alpha,colon-specific antigen-p (CSAp), CEA(CEACAM5), CEACAM6, c-met, DAM,EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folatereceptor, G250 antigen, GAGE, gp100, GPA33, GROB, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunit, HER2/neu, HER3, HMGB-1,hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ,IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, IL-33, insulin-likegrowth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, L1CAM, Le-Y,LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3,MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MICA, MICB, MIP-1A,MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2,MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, placental growthfactor, p53, PLAGL2, prostate acid phosphatase, PSA, PRAME, PSMA, P1GF,ILGF, ILGF-1R, IL-6, IL-25, ROR-1, RS5, RANTES, T101, SAGE, 5100,survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-alpha,Tn antigen, Thomson-Friedrich antigen, tumor necrosis antigen, TROP-2,VEGFA, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factorC3, C3a, C3b, C5a, C5, angiogenesis marker, bc1-2, bc1-6, Kras, cMET,oncogene product, HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, meticillin-resistant Staphylococcus aureus, Legionellapneumophila, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Rabies virus, Influenza virus,Cytomegalovirus, Type I herpes simplex virus, type II herpes simplexvirus, human serum parvo-like virus, respiratory syncytial virus,varicella-zoster virus, hepatitis B virus, hepatitis C virus, measlesvirus, adenovirus, human T cell leukemia virus, Epstein-Barr virus,murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbisvirus, lymphocyte choriomeningitis virus, Wart virus, Blue tongue virus,Sendai virus, Cat leukemia virus, Reovirus, Poliovirus, Simian virus 40,mouse mammary tumor virus, Dengue fever virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, preferably anyone of CD19, CD20, CD22, CD30, CD33, CD38, CD123, Muc1, Muc16, HER2,HERS, EGFRvIII, VEGFA, CEA, GPA33, GP100, ANG2, L1CAM, ROR-1, CS1, MICAor MICB.

Preferably, the antigen expressed on the effector T cell is any one ofADAM17, CD2, CD3, CD4, CD5, CD6, CD8, CD11a, CD11b, CD14, CD16, CD16b,CD25, CD28, CD30, CD32a, CD40, CD40L, CD44, CD45, CD56, CD57, CD64,CD69, CD74, CD89, CD90, CD137, CD177, CEACAM6, CEACAM8, HLA-DRa chain,KIR, LSECtin or SLC44A2, preferably any one of CD2, CD3, CD4, CD5, CD6,CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 or CD90.

In a second aspect, the present invention also provides a method ofproducing a bispecific antibody as described in the first aspect of theinvention comprising connecting the nanomaterial to the first antibodymoiety and the second antibody moiety.

The method for producing a bispecific antibody according to the presentinvention comprises the steps of:

(1) preparation, collection and activation of a nanomaterial;(2) connecting the nanomaterial obtained in step (1) with a mixture ofthe first antibody moiety and the second antibody moiety.

In the step (1) of the invention, the preparation of the nanomaterialcomprises: dissolving the nanomaterial completely by a solvent, stirringand adding water to form a uniform emulsion.

Preferably, the nanomaterial is any one of polylactic acid-glycolicacid, polylactic acid, polycaprolactone, polybutylene glycol succinate,polyaniline, polycarbonate, glycolide-lactide copolymer orglycolide-caprolactone copolymer, or a mixture thereof.

Preferably, the solvent is any one of acetone, butanone, methanol,ethanol or isopropanol, or a mixture thereof.

Preferably, the collection of the nanomaterial comprises: collecting theprepared nanomaterial by centrifugation, and then washing thenanomaterial by resuspending in deionized water twice.

Preferably, the activation of the nanomaterial comprises: activating thenanomaterial for 0.5-5 hours by using a mixed solvent of 1-10 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS) andN-hydroxysuccinimide (NHS) at room temperature.

In the step (2) of the invention, the connection comprises: collectingthe activated nanomaterial by centrifugation, and then washing thenanomaterial once with the connecting reaction solution, adding amixture of the first antibody moiety and the second antibody moiety tobe connected in an equal volume into the connecting reaction solution,and then resuspending the nanomaterial with the connecting reactionsolution containing the first antibody moiety and the second antibodymoiety and conducting the connecting reaction for 0.5-5 hours at roomtemperature. After the reaction, the nanomaterial is collected bycentrifugation. The nanomaterial is washed twice in Dulbecco's phosphatebuffer saline (D-PBS), and then resuspended in D-PBS and stored at 4° C.

The method for producing bispecific antibodies described hereincomprises the following steps:

(1) Preparation of nanomaterials: The nanomaterial is completelydissolved in acetone at a concentration of 5 to 30 mg/mL, and thesolution of the nanomaterial with acetone is added to the deionizedwater in 1:4 v/v of acetone and deionized water with magnetic stirred at500 to 1500 rpm/min, to form a uniform emulsion and then continue tostir until the volatilization of acetone;(2) Collection of nanomaterials: collecting the prepared nanomaterialsby centrifugation at 8000˜15000 rpm/min, and then washing thenanomaterials by resuspending in deionized water twice;(3) Activation of nanomaterials: activating the nanomaterials by using amixed solvent of 1-10 mg/mL 1-ethyl-(3-dimethylaminopropyl)carbodiimidehydrochloride and N-hydroxysuccinimide at room temperature for 0.5˜5hours;(4) Connection of nanomaterials with antibodies: collecting theactivated nanomaterials by centrifugation, and then washing thenanomaterials once with 0.1 M D-PBS at pH=8.0, and adding a mixture ofthe first antibody moiety and the second antibody moiety to be connectedin an equal volume into the connecting reaction solution, and thenresuspending the nanomaterials with the connecting reaction solutioncontaining the first antibody moiety and the second antibody moiety andconducting the connecting reaction for 0.5-5 hours at room temperature.After the reaction, the nanomaterials are collected by centrifugation.The nanomaterials are washed twice in D-PBS, and then resuspended inD-PBS and stored at 4° C.

In a third aspect, the present invention also provides the use of thebispecific antibody as described in the first aspect in the manufactureof a medicament for the treatment, prevention or diagnosis of a tumor.The tumor includes, but not limited to, liver cancer, non-small celllung cancer, small cell lung cancer, adrenocortical carcinoma, acute(chronic B) lymphocytoma, myeloma, prostate cancer, breast cancer,esophageal cancer, gastric cancer, colorectal cancer, cervical cancer,kidney cancer, bladder cancer and lymphoma.

In a fourth aspect, the present invention also provides the bispecificantibody according to any one of claims 1 to 3 for use in the treatmentor prevention of a tumor. The tumor includes, but not limited to, livercancer, non-small cell lung cancer, small cell lung cancer,adrenocortical carcinoma, acute (chronic B) lymphocytoma, myeloma,prostate cancer, breast cancer, esophageal cancer, gastric cancer,colorectal cancer, cervical cancer, kidney cancer, bladder cancer andlymphoma.

In a fifth aspect, the present invention provides a method of treating atumor comprising administering to a subject the bispecific antibody ofthe invention. The tumor includes, but not limited to, liver cancer,non-small cell lung cancer, small cell lung cancer, adrenocorticalcarcinoma, acute (chronic B) lymphocytoma, myeloma, prostate cancer,breast cancer, esophageal cancer, gastric cancer, colorectal cancer,cervical cancer, kidney cancer, bladder cancer and lymphoma.

Compared with the prior art, the invention has at least the followingbeneficial effects:

(1) The bispecific antibody according to present invention can be morequickly, simply and practically produced as compared to the existingbispecific antibodies which are expressed by biological methods. Thebispecific antibody according to present invention can be connecteddirectly to the monoclonal antibodies approved on the market. Becausethe raw materials to be used are all clinically approved on the market,the bispecific antibody prepared according to the invention can quicklyenter clinical use;(2) Compared with immune cell therapy via the genetically-modifiedchimeric antigen receptor T cell (CAR-T), the bispecific antibody of thepresent invention has disadvantages such as biodegradability, no generecombination, low side effect, high safety, low cost, capability tocombine respective specific antibodies for various tumor cells, ease ofuse, etc. According to present invention, a similar effect on targetingand efficiently killing cancer cells can be achieved by administratingthe bispecific antibody preparation of the present invention to asubject while returning the CTL cells induced in vitro to the subjectvia infusion. The resulted side effects are lower than CAR-T therapy;(3) All of the materials of the present invention can be degraded intonon-toxic and harmless products in the human body and can be degradedand metabolized shortly. Accordingly, the present invention is saferthan CAR-T.

DESCRIPTION OF DRAWINGS

FIG. 1 shows assembling of the bispecific antibody of Example 1.

FIG. 2 shows the result of the cell killing experiments of thebispecific antibody of Example 1.

FIGS. 3-9 show the ability of other bispecific antibodies to kill tumorcells.

FIG. 10 shows the efficacy of a bispecific antibodyanti-CD3-PLGA-anti-MUC1 on inhibiting tumor in vivo.

FIG. 11 shows the curve of laboratory animal mortality for antitumorexperiments in vivo.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

The invention is described in details through the Examples, withreference to the accompanying drawings.

EXAMPLES Example 1

FIG. 1 shows assembling of the bispecific antibody of this Example. Theprocess comprises the following specific steps:

(1) Preparation of PLGA nanoparticles: PLGA was completely dissolved toa concentration of 5 mg/mL using acetone, and the solution of PLGA andacetone was added into deionized water in a volume ratio of 1:4 ofacetone and deionized water with magnetic stirring at 1000 rpm/min, toform a uniform emulsion, and then continue to stir until volatilizationof acetone;(2) Collection of PLGA nanoparticles: collecting the preparednanoparticles with larger particle size by centrifugation at 8000rpm/min for 10 min; then collecting the prepared nanoparticles withsmaller particle size by centrifugation at 15000 rpm/min for 10 min, andthen resuspended in deionized water respectively, and repeatedly washingthe nanomaterials twice; nanoparticles with larger particle size andnanoparticles with smaller particle size were carried out as follows;(3) Activation of PLGA nanoparticles: using a mixed solvent of 5 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature to activate PLGA nanopoarticlesfor 1 h;(4) Connecting PLGA nanoparticles to antibodies: collecting theactivated nanomaterials by centrifugation, and then washing thenanomaterial once with 0.1 M D-PBS at pH=8.0, and adding a mixture ofCD3 and Muc1 monoclonal antibodies to be connected in an equal volumeinto the connecting reaction solution, and then resuspending thenanomaterial with the connecting reaction solution containing the firstantibody moiety and the second antibody moiety and conducting theconnecting reaction for 0.5 h at room temperature. After the reaction,the nanomaterial is collected by centrifugation. The nanomaterial iswashed twice in D-PBS, and then resuspended in D-PBS and stored at 4° C.

Example 2

The bispecific antibody of Example 1 was added to T cell and cancer cellMCF-7 killing experimental system to detect the ability of thebispecific antibody to enhance the ability of T cell to kill cancercell, in which T cell: MCF-7=4:1, the reaction time is 8 h, in which thecontrol group is the same amount of nanoparticles (not connected to anyantibody). The results are expressed as the average±standard deviationof three independent experiments. The evaluation results are shown inFIG. 2, in which * indicates a significant difference in T test.

The A in FIG. 2 represents bispecific antibodies connected tonanoparticles with a larger particle size (i.e., particles collected bycentrifugation at 8000 rpm/min for 10 min), while B representsbispecific antibodies connected to nanoparticles with a smaller particlesize (i.e., particles not collected by centrifugation at 8000 rpm/minfor 10 min but collected by centrifugation at 15,000 rpm/min for 10min).

It can be seen from FIG. 2 that the killing rate of the control group is7.9%; the killing rate of the group of the bispecific antibodiesconnected to nanoparticles with a larger particle size is 24.5%; thekilling rate of the group of the bispecific antibodies connected tonanoparticles with a smaller particle size is 40.7%.

Example 3

Using PLA as nanomaterial, bispecific antibodies capable of beingcombined with immune cells to enhance the targeting tumor killingability were prepared. The preparation process is as follows:

(1) Preparation of PLA nanoparticles: PLA was completely dissolved to aconcentration of 15 mg/mL using acetone, and the solution of PLA andacetone was added into deionized water in a volume ratio of 1:4 ofacetone and deionized water with magnetic stirring at 500 rpm/min, toform a uniform emulsion, and then continue to stir until volatilizationof acetone;(2) Collection of PLA nanoparticles: collecting the preparednanoparticles by centrifugation at 8000 rpm/min, and then resuspended indeionized water, and repeatedly washing the nanomaterials twice;(3) Activation of PLA nanoparticles: using a mixed solvent of 1 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature to activate PLA nanopoarticlesfor 0.5 h;(4) Connecting PLA nanoparticles to antibodies: collecting the activatednanomaterials by centrifugation, and then washing the nanomaterials oncewith 0.1 M D-PBS at pH=8.0, and adding a mixture of CD3 and CD19monoclonal antibodies to be connected in an equal volume into theconnecting reaction solution, and then resuspending the nanomaterialswith the connecting reaction solution containing the first antibodymoiety and the second antibody moiety and conducting the connectingreaction for 5 h at room temperature. After the reaction, thenanomaterials are collected by centrifugation. The nanomaterials arewashed twice in D-PBS, and then resuspended in D-PBS and stored at 4° C.

Example 4

Using PCL as nanomaterials, bispecific antibodies capable of beingcombined with immune cells to enhance the targeting tumor killingability was prepared. The preparation process is as follows:

(1) Preparation of PCL nanoparticles: PCL was completely dissolved to aconcentration of 30 mg/mL using acetone, and the solution of PCL andacetone was added into deionized water in a volume ratio of 1:4 ofacetone and deionized water with magnetic stirring at 1500 rpm/min, toform a uniform emulsion, and then continue to stir until volatilizationof acetone;(2) Collection of PCL nanoparticles: collecting the preparednanoparticles by centrifugation at 15000 rpm/min, and then resuspendedin deionized water, and repeatedly washing the nanomaterials twice;(3) Activation of PCL nanoparticles: using a mixed solvent of 10 mg/mL1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide at room temperature to activate PCL nanopoarticlesfor 5 h;(4) Connecting PCL nanoparticles to antibodies: collecting the activatednanomaterials by centrifugation, and then washing the nanomaterials oncewith 0.1 M D-PBS at pH=8.0, and adding a mixture of CD3 and CD20monoclonal antibodies to be connected in an equal volume into theconnecting reaction solution, and then resuspending the nanomaterialswith the connecting reaction solution containing the first antibodymoiety and the second antibody moiety and conducting the connectingreaction for 2.5 h at room temperature. After the reaction, thenanomaterials are collected by centrifugation. The nanomaterials arewashed twice in D-PBS, and then resuspended in D-PBS and stored at 4° C.

Example 5

The ability of PLGA-connected anti-CD3 and anti-MUC1 bispecificantibodies (anti-CD3-PLGA-anti-MUC1) to kill other tumor cells wasevaluated.

Specifically, 5000 target cells were cultured in each well for 12 h in96-well plates and then the original medium was discarded. The densityof DC-CIK cells was adjusted by X-vivo 15 medium without cytokine,resulting in that DC-CIK cell number in a volume of 100 μl was 4 timeslarger than the target cells (effect-target ratio is 4:1). 100 μlsuspension of DC-CIK cells was added in a cancer cell culture plate, and10 μl prepared bispecific antibody (bispecific antibody preparationcontent: 0.2 mg, with a total amount of monoclonal antibodies 0.2 μg)was added, incubated for 8 h in an incubator, and then CCK-8 reagent wasadded, incubated according to the reagent instructions. The absorbanceat 450 nm was measured using a microplate reader. Statistical analysisof the data was performed, according to the following formula tocalculate killing rate of DC-CIK cells on cancer cells.

Killing rate=[1−(experimental group−effect control group)/(targetcontrol group−blank control group)]×100%

The blank control group represents the added medium; the target controlgroup represents the added target cell+medium; the effect control grouprepresents the added the effector cell+medium; the experimental grouprepresents the effector cell+target cell+medium+bispecific antibody.

The results are shown in Table 1 and FIG. 3.

TABLE 1 The ability of anti-CD3-PLGA-anti-MUC1 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group HKC 9.66% 13.21% A549  7.59% 87.35% HcpG2 10.67% 82.44% HT29 13.84% 91.27%AGS 17.56% 92.17% Hela 13.57% 87.54% 5637 11.64% 82.51% OS-RC-2 12.24%87.57% Note: HKC - human embryo kidney epithelial cells; A549 - humannon-small cell lung cancer cells; HepG2 - human liver cancer cells;HT29 - human colon cancer cells; AGS - human gastric adenocarcinomacells; Hela - human cervical cancer cells; 5637 - human bladder cancercells; OS-RC-2 - human kidney cancer cells.

Example 6

Using the processes method as described above, with PLGA, PLA or PCL asnanomaterials, anti-CD3 and anti-CD33 bispecific antibodies connectedvia nanoparticles, i.e. anti-CD3-PLGA-anti-CD33, anti-CD3-PLA-anti-CD33and anti-CD3-PCL-anti-CD33, were prepared. The ability of the bispecificantibodies of the invention to kill tumor cells was evaluated accordingto the method described in Example 5.

The results are shown in Table 2 and FIG. 4.

TABLE 2 The ability of anti-CD3-PLGA-anti-CD33 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group Raji 8.59% 85.91% RAMOS 13.67% 83.36% Note: Raji - black Burkitt lymphomacells; RAMOS - human B lymphocytoma cells.

The bispecific antibodies CD3-PLA-CD33 and CD3-PCL-CD33 both showed akilling rate of more than 83% for black Burkitt lymphoma cells and humanB lymphocytoma cells.

Example 6

Using the similar processes as described above, with PLGA, PLA or PCL asnanomaterials, anti-CD8 and anti-MUC1 bispecific antibodies connectedvia nanoparticles, i.e. anti-CD8-PLGA-anti-MUC1, anti-CD8-PLA-anti-MUC1and anti-CD8-PCL-anti-MUC1, were prepared. The ability of the bispecificantibodies of the invention to kill tumor cells was evaluated accordingto the method described in Example 5.

The results are shown in Table 3 and FIG. 5.

TABLE 3 The ability of anti-CD8-PLGA-anti-MUC1 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group HKC 9.66% 13.21% A549  7.59% 87.35% HepG2 10.67% 82.44% HT29 13.84% 91.27%MCF-7  8.62% 87.23% AGS 17.56% 92.17% Hela 13.57% 87.54% 5637 11.64%82.51% OS-RC-2 12.24% 87.57% Note: HKC - human embryo kidney epithelialcells; A549 - human non-small cell lung cancer cells; HepG2 - humanliver cancer cells; HT29 - human colon cancer cells; AGS - human gastricadenocarcinoma cells; Hela - human cervical cancer cells; 5637 - humanbladder cancer cells; OS-RC-2 - human kidney cancer cells.

The bispecific antibodies anti-CD8-PLA-anti-MUC1 andanti-CD8-PCL-anti-MUC1 both showed a killing rate of more than 95% forhuman non-small cell lung cancer cells and human gastric adenocarcinomacells; both showed a killing rate of more than 92% for human coloncancer cells and human kidney cancer cells; both showed a killing rateof nearly 90% for human breast cancer cells, human cervical cancer cellsand human bladder cancer cells; showed a killing rate of about 80% forhuman liver cancer cells.

Example 7

Using the similar process as described above, other bispecificantibodies connected via nanoparticles, i.e. anti-CD3-PLGA-anti-CD19,anti-CD3-PLGA-anti-CD20, anti-CD3-PLGA-anti-her2 were prepared. Theability of the bispecific antibodies to kill tumor cells was evaluated.The results are shown in Tables 4-7 and FIGS. 6-9.

TABLE 4 The ability of anti-CD3-PLGA-anti-CD19 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group Raji 8.59% 82.91% RAMOS 13.67% 89.36% Note: Raji - black Burkitt lymphomacells; RAMOS - human B lymphocytoma cells.

TABLE 5 The ability of anti-CD3-PLGA-anti-CD20 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group Raji 8.59% 92.71% RAMOS 13.67% 88.27% Note: Raji - black Burkitt lymphomacells; RAMOS - human B lymphocytoma cells.

TABLE 6 The ability of anti-CD3-PLGA-anti-her2 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group MCF-719.88% 63.84% Note: MCF-7 - human breast cancer cells.

TABLE 7 The ability of anti-CD3-PLGA-anti-CD38 to kill tumor cellsKilling rate Cancer cell Effect control group Experimental group Raji28.55% 87.78% Note: Raji - black Burkitt lymphoma cells.

Example 8. Anti-Tumor Experiment In Vivo

The anti-tumor effect of the bispecific antibodies of the presentinvention were evaluated. Specifically, healthy Balb-c nude mice (cleangrade, female, four weeks old, 18-22 g in weight, purchased from theGuangdong Provincial Medicine Laboratory Animal Center) were inoculatedwith human lung adenocarcinoma cell A549 at armpit. After 3-4 weeks ofinoculation, 30 nude mice with tumor diameter of about 0.5×0.5 cm wererandomly divided into three groups for the experiment.

At the time of day 1, 3, 5, all mice were administered once via thetail-intravenous injection. 1×10⁶ DC-CIK cells were injected each timein the DC-CIK group; 1×10⁶ DC-CIK cells and 0.4 mg (a total amount ofmonoclonal antibody 0.4 μg) bispecific antibody anti-CD3-PLGA-anti-MUC1were each time injected in the DC-CIK+bispecific antibody group; thesame volume of saline was injected in the control group.

The tumor volume was recorded from the first day. Calculate theinhibitory rate of the bispecific antibodies against tumors.

After the death of all the animals in the control group and the DC-CIKalone treatment group, the experiment was terminated. The total time ofobservation was 128 days.

The average tumor volume of each group is shown in FIG. 10. The resultsshowed that the inhibitory rate of the bispecific antibodyanti-CD3-PLGA-anti-MUC1 against tumors was 88.73%, and the tumors of 60%of the animals (6/10) completely disappeared.

In terms of survival time, treatment with DC-CIK alone and treatmentwith DC-CIK+bispecific antibody both provided significant survivalbenefit when compared to untreated mice (FIG. 11). All the untreatedmice died before day 105. All mice in the DC-CIK only treatment groupdied before day 128, while 6 mice still survived in theDC-CIK+bispecific antibody treatment group.

As can be seen from the above Examples, the bispecific antibodies of thepresent invention have an improved ability to kill cancer cells by Tcells, can be combined with the immune cells to enhance tumor killingability, and have advantages such as less side effects, higher safety,lower cost, easy to use and so on as compared to CAR-T immune celltherapy.

The Applicant declares that the present invention illustrates theprocesses of the present invention via the above-described Examples, butthe invention is not limited to the above-described process steps, i.e.it is not meant that the present invention must be carried out inaccordance with the above-described process steps. It will be apparentto those skilled in the art that any improvements to the presentinvention, equivalents of the materials selected for use in the presentinvention, addition of auxiliary ingredients, selection of specificways, etc., are within the disclosure and protection scope of thepresent invention without departing from the idea, spirit and scope ofthe invention.

1.-17. (canceled)
 18. A bispecific antibody, comprising a first antibodymoiety that binds to an antigen expressed on an effector T cell and asecond antibody moiety that binds to an antigen expressed on a cancercell, wherein the first antibody moiety and the second antibody moietyare connected by a biodegradable nanomaterial, wherein the antigenexpressed on an effector T cell is not CD40.
 19. The bispecific antibodyaccording to claim 18, wherein the nanomaterial is any one of polylacticacid-glycolic acid, poly lactic acid, polycaprolactone, polybutyleneglycol succinate, polyaniline, polycarbonate, glycolide-lactidecopolymer or glycolide-caprolactone copolymer, or a mixture thereof. 20.The bispecific antibody according to claim 18, wherein the antigenexpressed on the cancer cell is selected from the group consisting ofCD19, CD20, CD22, CD30, CD33, CD123, Muc1, Muc16, HER2, HER3, EGFRvIII,VEGFA, CEA, GPA33, GP100, ANG2, L1CAM, ROR-1, CS1, MICA and MICB; theantigen expressed on the effector T cell is selected from the groupconsisting of CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40L,CD44, CD45, CD69, and CD90.
 21. The bispecific antibody according toclaim 18, wherein the antigen expressed on the cancer cell is selectedfrom the group consisting of carbonic anhydrase IX, alpha-fetoprotein,alpha-actinin-4, A3, A33 antibody-specific antigen, ANG2, ART-4, B7, Ba733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21,CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70,CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138,CD147, CD154, CDC27, CDK-4/m, CDKN2A, CS1, CXCR4, CXCR7, CXCL12,HIF-1alpha, colon-specific antigen-p (CSAp), CEA(CEACAM5), CEACAM6,c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3,folate receptor, G250 antigen, GAGE, gp100, GPA33, GROB, HLA-DR, HM1.24,human chorionic gonadotropin (HCG) and its subunit, HER2/neu, HERS,HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R,IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, IL-33,insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4,L1CAM, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF),MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MICA, MICB,MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16,MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, placentalgrowth factor, p53, PLAGL2, prostate acid phosphatase, PSA, PRAME, PSMA,P1GF, ILGF, ILGF-1R, IL-6, IL-25, ROR-1, RS5, RANTES, T101, SAGE, S100,survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-alpha,Tn antigen, Thomson-Friedrich antigen, tumor necrosis antigen, TROP-2,VEGFA, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factorC3, C3a, C3b, C5a, C5, angiogenesis marker, bc1-2, bc1-6, Kras, andcMET, and the antigen expressed on the effector T cell is selected fromthe group consisting of ADAM17, CD2, CD3, CD4, CD5, CD6, CD8, CD11a,CD11b, CD14, CD16, CD16b, CD25, CD28, CD30, CD32a, CD40L, CD44, CD45,CD56, CD57, CD64, CD69, CD74, CD89, CD90, CD137, CD177, CEACAM6,CEACAM8, HLA-DRa chain, KIR, LSECtin or SLC44A2.
 22. A bispecificantibody, comprising a first antibody moiety that binds to an antigenexpressed on an effector T cell and a second antibody moiety that bindsto an antigen expressed on a cancer cell, wherein the first antibodymoiety and the second antibody moiety are connected by a biodegradablenanomaterial, and wherein the antigen expressed on the effector T cellis CD3 and the antigen expressed on the target cell is Muc1, or whereinthe antigen expressed on the effector T cell is CD3 and the antigenexpressed on the target cell is CD19, or wherein the antigen expressedon the effector T cell is CD3 and the antigen expressed on the targetcell is CD20, or wherein the antigen expressed on the effector T cell isCD3 and the antigen expressed on the target cell is CD33, or wherein theantigen expressed on the effector T cell is CD3 and the antigenexpressed on the target cell is Her2, or wherein the antigen expressedon the effector T cell is CD8 and the antigen expressed on the targetcell is Muc1, or wherein the antigen expressed on the effector T cell isCD3 and the antigen expressed on the target cell is CD38.
 23. Thebispecific antibody according to claim 22, wherein the nanomaterial isany one of polylactic acid-glycolic acid, polylactic acid,polycaprolactone, polybutylene glycol succinate, polyaniline,polycarbonate, glycolide-lactide copolymer or glycolide-caprolactonecopolymer, or a mixture thereof.
 24. A method for producing a bispecificantibody according to claim 18, which comprises connecting thenanomaterial to the first antibody moiety and to the second antibodymoiety.
 25. The method according to claim 24, which comprises the stepsof: (1) preparation, collection and activation of the nanomaterial; (2)connecting the nanomaterial obtained in step (1) with a mixture of thefirst antibody moiety and the second antibody moiety.
 26. The methodaccording to claim 25, wherein the nanomaterial is any one of polylacticacid-glycolic acid, poly lactic acid, polycaprolactone, polybutyleneglycol succinate, polyaniline, polycarbonate, glycolide-lactidecopolymer or glycolide-caprolactone copolymer, or a mixture thereof; andthe nanomaterial dissolved in a solvent selected from acetone, butanone,methanol, ethanol or isopropanol or a mixture thereof.
 27. A method oftreating a tumor in a subject comprising administering to the subject abispecific antibody according to claim
 18. 28. The method according toclaim 27, wherein the tumor is selected from the group consisting ofliver cancer, non-small cell lung cancer, small cell lung cancer,adrenocortical carcinoma, acute (chronic B) lymphocytoma, myeloma,prostate cancer, breast cancer, esophageal cancer, gastric cancer,colorectal cancer, cervical cancer, kidney cancer, bladder cancer andlymphoma.
 29. A method for producing a bispecific antibody according toclaim 22, which comprises connecting the nanomaterial to the firstantibody moiety and to the second antibody moiety.
 30. The methodaccording to claim 29, which comprises the steps of: (1) preparation,collection and activation of the nanomaterial; (2) connecting thenanomaterial obtained in step (1) with a mixture of the first antibodymoiety and the second antibody moiety.
 31. The method according to claim30, wherein the nanomaterial is any one of polylactic acid-glycolicacid, poly lactic acid, polycaprolactone, polybutylene glycol succinate,polyaniline, polycarbonate, glycolide-lactide copolymer orglycolide-caprolactone copolymer, or a mixture thereof; and thenanomaterial dissolved in a solvent selected from acetone, butanone,methanol, ethanol or isopropanol or a mixture thereof.
 32. A method oftreating a tumor in a subject comprising administering to the subject abispecific antibody according to claim
 22. 33. The method according toclaim 32, wherein the tumor is selected from the group consisting ofliver cancer, non-small cell lung cancer, small cell lung cancer,adrenocortical carcinoma, acute (chronic B) lymphocytoma, myeloma,prostate cancer, breast cancer, esophageal cancer, gastric cancer,colorectal cancer, cervical cancer, kidney cancer, bladder cancer andlymphoma.